Interaction of PINK1 with nucleotides and kinetin

The ubiquitin kinase PINK1 accumulates on damaged mitochondria to trigger mitophagy, and PINK1 loss-of-function mutations cause early onset Parkinson’s disease. Nucleotide analogs such as kinetin triphosphate (KTP) were reported to enhance PINK1 activity and may represent a therapeutic strategy for the treatment of Parkinson’s disease. Here, we investigate the interaction of PINK1 with nucleotides, including KTP. We establish a cryo-EM platform exploiting the dodecamer assembly of Pediculus humanus corporis (Ph) PINK1 and determine PINK1 structures bound to AMP-PNP and ADP, revealing conformational changes in the kinase N-lobe that help establish PINK1’s ubiquitin binding site. Notably, we find that KTP is unable to bind PhPINK1 or human (Hs) PINK1 due to a steric clash with the kinase “gatekeeper” methionine residue, and mutation to Ala or Gly is required for PINK1 to bind and use KTP as a phosphate donor in ubiquitin phosphorylation and mitophagy. HsPINK1 M318G can be used to conditionally uncouple PINK1 stabilization and activity on mitochondria.

Enhancing mitophagy by pharmacologically increasing PINK1 activity has been considered as a potential strategy to treat PD (24,25).In a key study, an analog of ATP, kinetin triphosphate (KTP), was reported to be used by PINK1 with greater efficiency than ATP and restored the activity of a PINK1 EOPD mutant (26).While KTP itself is impermeable to the cell membrane and therefore of limited therapeutic value, its membrane permeable precursor, kinetin, can be intracellularly metabolized into KTP and appeared to accelerate PINK1/Parkin mitophagy (26,27).However, it remains unclear whether the PINK1 activating effect of kinetin is mediated through KTP or whether an alternate mechanism is involved (28).Regardless, these compounds demonstrate a considerable therapeutic potential, and it is important to understand the molecular basis underlying their activities.
Here, we investigate PINK1's interaction with the nucleotides adenylyl-imidodiphosphate (AMP-PNP) and adenosine 5′-diphosphate (ADP) and with the reported PINK1 activators KTP and kinetin.We exploit a PhPINK1 dodecamer as a platform to determine nucleotidebound PINK1 structures by cryo-electron microscopy (cryo-EM).While structures of AMP-PNP-bound and ADP-bound PhPINK1 reveal nucleotide-induced conformational changes in the PINK1 N-lobe, we failed to detect any binding between PINK1 and KTP.Instead, KTP binding is blocked by PINK1's gatekeeper residue, which is analogous to many other kinases.Mutation of the Met gatekeeper residue to smaller residues enables KTP binding, and mutation to a Gly switches PINK1's nucleotide preference from ATP to KTP, which inactivates PINK1 in cells.Mutated PINK1 can now be activated by treatment of cells with kinetin and be used as a conditional activator of gatekeepermutated PINK1, allowing us to uncouple PINK1 stabilization and PINK1 activity in mitophagy settings.

A PhPINK1 dodecamer enables atomic-resolution cryo-EM structures of PINK1 bound to nucleotides
We recently reported that a PhPINK1 construct (residues 115 to 575) in its unphosphorylated state could assemble into a homo-dodecameric complex, enabling structural determination of the complex by cryo-EM (12).However, these structures of PhPINK1 did not contain nucleotides and were obtained with kinase inactive mutants or had undergone Cys-cross-linking procedures.The arrangement of molecules within the dodecamer (comprising six PhPINK1 dimers) provided key insights into PINK1 activation, and the PhPINK1 dodecamer itself, with open and unobstructed ATP binding sites (Fig. 1A), was the ideal platform to determine whether and how nucleotide binding alters PINK1 conformation.We therefore sought to determine structures of wild-type (WT) PhPINK1, without cross-linking, bound to ADP or the non-hydrolyzable ATP analog AMP-PNP.
Thermal shift assays confirmed that ADP, ATP, and AMP-PNP, each in the presence of Mg 2+ , bind and stabilize monomeric PhPINK1 (fig.S1).To obtain WT PhPINK1 dodecamers for cryo-EM analysis, PhPINK1 was purified from bacteria in its monomeric and autophosphorylated form (20). Dephosphorylation using λphosphatase (λ-PP) induces oligomerization into dodecamers that could be isolated and purified (fig.S2A; see Materials and Methods).Phos-tag analysis, which resolves proteins according to phosphorylation status, confirmed that PhPINK1 was homogeneously dephosphorylated (fig.S2B), although a single phosphate remains on Thr 305 due to the adjacent Pro306 that prevents dephosphorylation by λ-PP (figs.S2B and 3C).The PhPINK1 dodecamer was left untreated or was incubated with AMP-PNP/Mg 2+ or ADP/Mg 2+ , before preparation of cryo-EM grids (Fig. 1B, fig.S2C, and Materials and Methods).Processing in cryoSPARC (29) yielded reconstructions of the sixfold symmetric dodecamer, which was subsequently locally refined using a single dimer (asymmetric unit) to resolutions of 2.75, 2.84, and 3.13 Å for the nucleotide-free, AMP-PNP-bound, and ADP-bound PhPINK1 dimers, respectively (Fig. 1C; fig.S2, C and D; and Table 1).Clear density could be observed for the bound nucleotides and Mg 2+ ions (Fig. 1C and fig.S3, A and B).The resolution of the maps enabled unambiguous modeling of PhPINK1 with each nucleotide.

Nucleotides induce conformational changes in the PINK1 N-lobe
The newly generated PhPINK1 structures revealed fresh insights into the interaction of nucleotides with PINK1 and unveiled notable conformational changes that couple nucleotide binding to previously observed conformational changes.The nucleotide-free WT PhPINK1 dimer was virtually indistinguishable from the nucleotide-free PhPINK1 D357A dimer that we determined previously (12).Both AMP-PNP and ADP bind the ATP binding site of PhPINK1 in the anticipated nucleotide binding mode and in a similar manner to TcPINK1 (Fig. 2A and fig.S3D) (13,22).Hydrophobic residues stemming from N-and C-lobes encapsulate the adenine and ribose groups (Fig. 2A).Two hydrogen bonds are made via the N 1 and N 6 nitrogens of adenine, which contact the backbone of Tyr 293 and Lys 291 , respectively, of the kinase hinge region (Fig. 2A).The phosphate groups of AMP-PNP and ADP extend toward the phosphoryl transfer center of the kinase and only indirectly bind the so-called P-loop (an extended Gly-rich βhairpin loop including the β1 and β2 strands) of the N-lobe (Fig. 2A).The phosphates are held in position by a series of electrostatic interactions with Lys 193 of the kinase "VAIK" motif (LAVK in PhPINK1) and two Mg 2+ ions, which themselves are positioned by several polar residues that include Asp 357 of the kinase "DFG" motif (Fig. 2A).Comparison of the nucleotide-free and nucleotide-bound states of PhPINK1 revealed prominent reorganization of the kinase P-loop upon nucleotide binding, which affects insertion-3 that eventually forms the ubiquitin binding site at the kinase N-lobe (Fig. 2B).In nucleotide-free (and unphosphorylated) PhPINK1, insertion-3 is only partially visible, but the ordered residues interact with and shield an otherwise exposed hydrophobic patch on the N-lobe, including Pro 164 , Ala 166 , and Val 172 of the P-loop (Fig. 2B and fig.S4A).This partially ordered conformation of insertion-3 is structurally incompatible with the fully ordered insertion-3 formed after PINK1 N-lobe phosphorylation that becomes the binding site for ubiquitin and Ubl substrates (Fig. 2C) (12,20).Binding of AMP-PNP or ADP to unphosphorylated PhPINK1 causes the P-loop to clamp onto the nucleotide.P-loop movement (by ~6 Å between the Cα's of Ile 165 ) involves multiple residues, including Val 173 of the β2 strand that forms part of the catalytic spine (C spine), which is completed upon interaction with the nucleotide (Fig. 2B) (30).
Two residues in the β1 strand, Ala 166 and Lys 167 , flip such that Ala 166 points its side chain into the ATP binding site, and Lys 167 points away from the ATP binding site (Fig. 2B).As a net result, clamping down of the P-loop generates additional space for insertion-3 to fold, although without phosphorylation, insertion-3 remains disordered, and several hydrophobic side chains on the N-lobe, including the P-loop, are exposed (Fig. 2B and fig.S4A).Also important is the observed flip in Lys 167 , which only in the nucleotide-bound or phosphorylated state of PINK1 can form interactions with both the ordered insertion-3, as well as with the substrate ubiquitin/Ubl (Fig. 2C) (12,20).
Together, these observations suggest an unexpected coupling between PINK1 nucleotide binding and the conformation of ubiquitinbinding insertion-3, via conformational rearrangements of the kinase P-loop.Given that phosphorylated PhPINK1 in its nucleotide-free state adopts both active and inactive conformations (12), it is tempting to speculate that ATP, in addition to its role as a phosphate donor, may contribute to stabilizing PINK1 in its active and ubiquitin bindingcompetent state.On the basis of conservation, this may be similar in human PINK1 (figs.S4B and S5; see Discussion).Intrinsic protein dynamics, confined localized changes, and low overall binding affinities make such conformational transitions difficult to assess biochemically.

Structures reveal a post-catalytic dimerized state of PINK1
Unexpectedly, we find that ADP-bound PhPINK1 is autophosphorylated at Ser 202 , most likely due to contaminating ATP in the ADP stock that we used (Fig. 2A and fig.S3, E to G).This additional phosphate group interacts with both Mg 2+ ions in the active site and is positioned ~5 Å from the βphosphate of the bound ADP (Fig. 2A and fig.S3G).The phosphorylated PhPINK1-ADP dimer represents the post-catalytic state of PINK1 immediately following phosphoryl transfer but before dimer dissociation [see (12)].In this situation, PhPINK1 molecules within the dimer remain entirely in their inactive conformation, with an extended αC helix and disordered insertion-3, contrasting the conformational shift observed in our previous structure of phosphorylated and cross-linked PhPINK1 dimer (12).A possible reason why the conformational change is not observed in this scenario could be that the high concentration of ADP in the sample sits snugly with phosphorylated Ser 202 (pSer 202 ) in the active site of a stable kinase dimer composition (fig.S3G).

PINK1 cannot use KTP due to a clash with a gatekeeper residue
We next attempted to investigate how the reported PINK1 activator KTP (Fig. 3A) interacts with PINK1 (26).Using thermal shift binding assays, we first tested whether KTP stabilizes PhPINK1 and observed that, unlike ATP, KTP does not stabilize PhPINK1 (Fig. 3B and fig.S6C).Next, in vitro ubiquitin phosphorylation assays showed that PhPINK1 could not phosphorylate ubiquitin when KTP was the sole nucleotide source (Fig. 3C).Given that the previous study reporting PINK1 activity with KTP was based on human PINK1 (26), we tested whether sequence differences between PhPINK1 and HsPINK1 (~40% kinase domain identity) may account for the inability of KTP to work with PhPINK1.To minimize these differences, we mutated the three differing residues within the PhPINK1 ATP binding site to HsPINK1 equivalent residues (V247I, M249V, and T356A), generating a humanized version of PhPINK1 (fig.S6A).Despite now harboring a HsPINK1-like ATP binding site, the PhPINK1 V247I/M249V/T356A triple mutant was neither stabilized nor showed ubiquitin kinase activity with KTP (fig.S6, B to D).
Next, we investigated HsPINK1 activity, using heavy membranes from oligomycin and antimycon (OA)-treated HeLa PINK1 −/− cells transiently expressing WT HsPINK1 that were incubated with recombinant ubiquitin in the presence of either ATP or KTP (see Materials and Methods).Incubation with ATP resulted in phosphorylation of ubiquitin at Ser 65 , as detected by a p Ser 65 ubiquitin antibody (Fig. 3D).While KTP incubation resulted in a faint phospho-ubiquitin band, a similarly faint band was also visible in the absence of any added nucleotide, indicating the presence of residual ATP in the crude heavy membrane preparation used for the assay (Fig. 3D).Together, these results indicate that PINK1 is unable to use KTP as a phosphate donor.
Why was PINK1 unable to use KTP in our experiments?KTP is an analog of ATP, defined by an additional furfuryl group covalently attached to the N 6 of the adenine ring (Fig. 3A).N 6 -substituted ATP analogs with bulky groups such as furfuryl typically cannot be accommodated by protein kinases due to a clash with a so-called gatekeeper residue at the back of the ATP binding site (31).It is possible that the conserved gatekeeper residue of PINK1 (Met 290 in PhPINK1 and Met 318 in HsPINK1) obstructs KTP (Fig. 3, E and F).Mutation of Met 290 in PhPINK1 to smaller Ala or Gly residues while affecting recombinant protein yield and stability (fig.S7) enabled the kinase to be stabilized by KTP and to use KTP as a phosphate donor in ubiquitin phosphorylation experiments (Fig. 3, G and H).These results were mirrored in HsPINK1 M318A and M318G mutants on mitochondria enriched from OA-treated HeLa cells (Fig. 3I).While mutant kinases were expressed at lower levels compared to WT PINK1 (Fig. 3I), the Gly mutation greatly diminished PINK1's ability to use ATP, consistent with the idea that the gatekeeper is also important for ATP binding (Fig. 3, H and I).However, both mutants were able to use KTP instead of ATP to generate phospho-ubiquitin.
Together, our results show that contrary to what has been suggested (26,32,33) PINK1 may not bind KTP or use it as a preferred phosphate donor in a direct ATP-competitive fashion.We could enable PINK1 to use KTP by mutating the gatekeeper Met residue in the PINK1 ATP binding site in insect and human PINK1.Switching PINK1's nucleotide preference from ATP to KTP was next exploited to decouple PINK1 stabilization from PINK1 activity.

Kinetin activates human PINK1 gatekeeper mutants in cells
We assessed the impact of the gatekeeper mutants M318A and M318G on HsPINK1 function in intact HeLa PINK1 −/− cells expressing YFP-Parkin that were transiently transfected with constructs encoding HsPINK1 variants.HsPINK1 WT accumulated in response to OA and generated phospho-ubiquitin, as expected (Fig. 4A).Accumulation of PINK1 was also observed for HsPINK1 M318A and M318G mutants, and proteasome inhibition induced similar accumulation of the 52-kDa presenilin-associated rhomboidlike protein (PARL)-cleaved PINK1 fragment, indicating that basal turnover of PINK1 variants was unimpaired (Fig. 4A).While a HsPINK1 M318A mutant phosphorylated ubiquitin at slightly reduced levels compared with WT, the M318G mutant generated phospho-ubiquitin at barely detectable levels (Fig. 4A), consistent with in vitro experiments (Fig. 3H).
Given the M318G mutant's preference for KTP over ATP, we wondered whether its inactivity in cells can be overcome by supplying KTP.However, direct KTP treatment is unfeasible as ATP analogs are unable to cross the cell membrane.Instead, the KTP precursor, kinetin, is membrane permeable and has been shown to be intracellularly metabolized into KTP (26).We therefore attempted to activate HsPINK1 M318G in cells with kinetin.Treatment with OA alone for 2 hours did not activate the M318G mutant, but cotreatment with 200 μM kinetin, but not adenine, led to substantial generation of phospho-ubiquitin (Fig. 4B).Increasing the duration of kinetin treatment to 24 hours did not increase the level of phospho-ubiquitin but instead hampered cell growth and/or survival (Fig. 4B).Kinetin did not enhance the activity of HsPINK1 WT, suggesting the effect of kinetin is specific to HsPINK1 M318G (fig.S8).These results are consistent with kinetin undergoing intracellular conversion into KTP, which then acts as a phosphate donor specifically for the HsPINK1 M318G gatekeeper mutant.We also generated cells stably expressing HsPINK1 M318G.As was observed in transient expression experiments, stably expressed HsPINK1 M318G accumulated in response to OA treatment and generated barely detectable levels of phospho-ubiquitin unless cotreated with kinetin (Fig. 4C).Phos-tag analysis further revealed that kinetin cotreatment increased PINK1 autophosphorylation and Parkin phosphorylation (Fig. 4C).

Kinetin-activated PINK1 M318G recruits Parkin to mitochondria
Because Parkin must be in proximity to OMM-stabilized PINK1 to become phosphorylated, our results suggested that kinetin-activated HsPINK1 M318G could induce translocation of Parkin to mitochondria and trigger mitochondrial clearance via mitophagy.To test this, we performed immunofluorescence using HeLa PINK1 −/− cells expressing YFP-Parkin and HsPINK1 to assess YFP-Parkin translocation to mitochondria.As anticipated, by the 1-hour time point of OA treatment, WT HsPINK1 had robustly recruited YFP-Parkin to mitochondria (Fig. 5).In contrast, HsPINK1 M318G did not recruit Parkin after 1 hour of OA treatment; however, when cotreated with kinetin, robust recruitment was observed at 1 hour of OA/kinetin treatment (Fig. 5).
Together, the results indicate that HsPINK1 M318G is functionally compromised to induce mitophagy in normal cells but now relies on a distinct nucleotide source, KTP, which is generated in situ from kinetin.We have hence established an orthogonal system to induce mitophagy, in which PINK1 stabilization and PINK1 activation are uncoupled.

DISCUSSION
In the first part of this study, we exploited established cryo-EM workflows to characterize the interaction of PhPINK1 with nucleotides.Our structures reveal localized conformational changes that occur upon nucleotide binding (Fig. 2).Crystal structures of nucleotide-free and AMP-PNP-bound TcPINK1 have been reported and compared previously (13,21,22), with a focus on understanding dimer formation and autophosphorylation reactions.We used cryo-EM to directly compare the nucleotide-bound and unbound states of PhPINK1, revealing conformational changes in the kinase P-loop and insertion-3 (fig.S5).Before nucleotide binding, insertion-3 shields a hydrophobic patch in the N-lobe, likely to maintain protein stability and prevent nonspecific interactions with other proteins before the activation of PINK1.Nucleotide binding is relayed via the flexible P-loop and opens the N-lobe to accommodate insertion-3; however, without phosphorylation, insertion-3 remains disordered.Such exposed state of PINK1 would be short-lived because subsequent trans-autophosphorylation of PINK1 would cause insertion-3 to reconfigure into its activated and ubiquitin binding conformation, thereby reshielding the hydrophobic patch (12,13,20).Furthermore, following autophosphorylation, insertion-3 remains dynamic and exchanges between folded and unfolded states, as we have previously resolved using three-dimensional (3D) variability analysis (12).Therefore, it is possible that ATP binding, in addition to it serving as a phosphate donor, helps to further stabilize insertion-3 in its folded and ubiquitin binding conformation, increasing the efficiency of ubiquitin binding and phosphorylation.
The ease with which the nucleotide-bound PhPINK1 structures could be solved means that the PhPINK1 dodecamer could now be used as a platform to understand the binding mechanism of other kinase-interacting molecules, such as PINK1-activating compounds.We had envisaged to use our platform to understand how KTP interacts with PINK1; however, we could not biochemically reproduce the reported PINK1-binding and activating properties of KTP (26).With our detailed structural understanding of PINK1, we define exactly why KTP cannot interact with PINK1: The PINK1 gatekeeper residue prevents the enlarged nucleobase to bind in the ATP binding pocket due to a steric clash between the KTP furfuryl group and the gatekeeper Met side chain.Mutation of the gatekeeper to the smaller residue such as Ala and Gly enables KTP to bind and activate PINK1.These results are consistent with numerous reports of kinases that require a gatekeeper mutation to use KTP (and KTP analogs such as KTPγS) as their phosphate source (34)(35)(36)(37)(38)(39).A proteome-wide, mass-spectrometry based study identified 26 putative KTP-binding kinases out of the 218 detected kinases in cell lysates (40).No endogenous human kinase bound KTP preferentially and even kinases that seemingly bound to KTP did not efficiently use KTP as a phosphate donor (40).That study did not analyze PINK1, possibly because of its very low abundance in unstimulated cells.Yet, our biochemical data presented here strongly suggest that WT PINK1 is unable to bind KTP or use it as a phosphate donor, let alone in a preferential manner.
The KTP precursor kinetin and its derivatives have been thought to activate PINK1 in cells by undergoing intracellular conversion into KTP to function as a phosphate donor (26,27,33).Our data now indicate that the underlying mechanism behind kinetininduced PINK1 activation, which has been reproduced by others, is unlikely to occur via KTP conversion and the provision of an improved nucleotide source to PINK1.A recent study reported that a divergent kinetin analog, MTK458, cannot be converted into a triphosphate form yet was able to activate PINK1 (28).How MTK458 acts on PINK1 will require further analysis and may also illuminate the role of kinetin in PINK1 biology.
Kinetin or MTK458 appear to work more efficiently in combination with subthreshold doses of depolarizing agents that are insufficient to trigger phospho-ubiquitin generation and mitophagy on their own (27,28).We used kinetin in combination with typical mitophagy-inducing doses of the depolarizing agent OA but did not detect kinetin-mediated amplification of HsPINK1 activity.Instead, we saw a remarkable sensitization to kinetin-mediated activation when the M318G gatekeeper mutation was introduced.The specificity of kinetin toward the M318G mutant strongly indicates that the mechanism underlying PINK1 activation in our case is via the conversion of kinetin into KTP, which then acts directly on PINK1 M318G via its enlarged ATP binding site.
Because HsPINK1 M318G accumulates on mitochondria upon depolarization yet remains almost completely inactive, kinetin may be used to specifically activate pre-accumulated HsPINK1 M318G, essentially decoupling PINK1 activity from stabilization of the protein through mitochondrial depolarization.A similar goal has been achieved previously using a temperature-sensitive mutant of PINK1 that becomes active when the temperature is lowered from 37° to 22°C (41).Our strategy, while not needing a temperature shift, requires the initial conversion of kinetin into KTP.On the basis of our immunofluorescence imaging experiments, conversion to KTP produces an effect on Parkin recruitment within 1 hour.If a more rapid conversion of kinetin is required, it may be possible to use kinetin riboside derivatives that reduce the number of conversion steps into KTP (33).We note that HsPINK1 M318G is not a kinase inactive mutant because it can function with ATP when expressed at high levels and subjected to extended OA treatments.Therefore, HsPINK1 M318G expression should be titrated to a level that induces minimal OA-induced Parkin recruitment and mitophagy while maintaining robust activation of kinase activity by kinetin.
The second kinase gatekeeper mutant that we used, HsPINK1 M318A, is active with both ATP and KTP and remains functional in the absence of kinetin.This mutant has been used previously for the purpose of sensitizing PINK1 to inhibition by PP1 analogs (such as small molecule compounds 1-NA-PP1 and 1-NM-PP1) that were specifically designed to inhibit gatekeeper-mutated kinases (42,43).It is likely that HsPINK1 M318G may also be inhibited by PP1 analogs, given that PP1 analogs inhibit many kinases harboring a Gly gatekeeper mutation (42).Therefore, kinetin and PP1 analogs, in combination with the HsPINK1 M318A and M318G gatekeeper mutants, may comprise a powerful chemical genetics toolkit for the activation or inhibition of PINK1 activity in cells in an experimentally controlled manner.

Protein expression and purification
All PhPINK1 (residues 115 to 575) constructs and λ-PP were expressed in E. coli Rosetta2 (DE3) pLacI cells (Novagen) and purified as described previously (12).To generate the WT PhPINK1 dodecamer for cryo-EM analysis, ~11 mg of purified WT PhPINK1 (residues 115 to 575), at a concentration of 15 μM, was dephosphorylated with 7.5 μM λ-PP in 25 mM tris (pH 8.5), 500 mM NaCl, 2 mM MnCl 2 , 5% (v/v) glycerol, 10 mM dithiothreitol (DTT) for 24 hours at 4°C.To promote PhPINK1 oligomerization, the concentration of NaCl was reduced by buffer exchange into 25 mM tris (pH 8.5), 150 mM NaCl, and 10 mM DTT using a HiPrep 26/10 Desalting column (Cytiva) and then incubated for 3 hours at 4°C.The resulting PhPINK1 dodecamer was purified by size exclusion chromatography (SEC) using a HiLoad 26/600 Superdex 200 pg column (Cytiva) in 25 mM tris (pH 8.5), 150 mM NaCl, and 10 mM DTT, and fractions corresponding to the dodecamer were pooled.Anion exchange chromatography was used to concentrate the dodecamer.Pooled fractions from SEC were applied to a Mono Q 5/50 GL column (Cytiva) in 25 mM tris (pH 8.5), 50 mM NaCl, and 10 mM DTT and eluted with a 0 to 50% linear gradient of 25 mM tris (pH 8.5), 1 M NaCl, and 10 mM DTT over 20 column volumes.The PhPINK1 dodecamer eluted at approximately 250 mM NaCl.The fraction containing the highest concentration of PhPINK1 (3.4 mg/ml) was diluted with 25 mM tris (pH 8.5) and 10 mM DTT to achieve a 150 mM NaCl concentration, resulting in a final PhPINK1 concentration of 1.9 mg/ml.The protein was then flash-frozen in liquid nitrogen and stored at −80°C.

Cryo-EM sample preparation and data collection
To generate the PhPINK1-nucleotide complexes, purified PhPINK1 dodecamer (1.9 mg/ml) was incubated with 10 mM AMP-PNP or ADP and 10 mM MgCl 2 for 10 to 25 min before vitrification.The nucleotide-free or nucleotide-bound dodecamers were dispensed onto glow discharged UltrAuFoil (Quantifoil GmbH, Germany) R1.2/1.3 holey specimen support ("grid") at 100% humidity, 4°C, and blotted for 4 s (nominal blot force of −1).Grids were then plunge frozen in liquefied ethane using a Vitrobot Mark IV (Thermo Fisher Scientific).Data were collected using a Titan Krios G4 microscope equipped with a Falcon 4 direct electron detector (Thermo Fisher Scientific) using a nominal magnification of ×96,000, corresponding to a pixel size at the detector of 0.808 Å.A total of 2746, 3094, and 2923 movies were captured for the nucleotide-free, AMP-PNP-bound, and ADP-bound PhPINK1 datasets, respectively.

Cryo-EM refinement and model building
Cryo-EM processing was performed in cryoSPARC (v4.2.1) (29).All PhPINK1 datasets were processed using a similar workflow, detailed in fig.S2C.All movies were patch-motion-corrected, and contrast transfer function (CTF) parameters were estimated using the patch CTF job.Templates were generated from blob picker performed on the pPhPINK1-ADP dataset and used to pick particles from all datasets using template picker.Particles were extracted and binned (downsampled) two times, and 2D classification was performed.Classes with any PINK1-like features were selected as "good" particles, and ab initio reconstruction was performed to generate an initial reconstruction of the PhPINK1 dodecamer.A second subset of classes containing particles of indistinct shapes was selected as "junk" particles, and ab initio reconstruction was performed to generate a volume for subsequent heterogeneous refinement.Three rounds of heterogeneous refinement were performed against the good and junk maps in C 1 to remove bad particles from the dataset.Reconstruction of the dodecamer was performed using homogeneous refinement in D 3 .A mask of the dimer was generated by zoning the map from homogenous refinement against a model of the PhPINK1 D357A dimer [Protein Data Bank (PDB): 7T4N] using UCSF ChimeraX (12,45), dilated, and soft-padded.Particles were symmetry expanded in D 3 , recentered, and locally refined using the dimer mask.For the AMP-PNP-bound and ADPbound PhPINK1 datasets, signal subtraction was performed before local refinement.3D variability analysis was performed solving for three modes, and the particles were clustered to give ~100,000 particles per cluster.Particles corresponding to the most homogeneous and complete cluster were then locally refined to give the final dimer reconstruction.
Model building was performed in Coot (v0.9.8.7) (46), and refinement was performed using real-space refinement in Phenix (v1.20.1-4487)(47).The PhPINK1 D357A dimer (PDB: 7T4N) (12) was used as the initial model and was docked into the density of the nucleotide-free PhPINK1 dimer using UCSF ChimeraX (v1.6.1)(45).After a round of model building in Coot and refinement in Phenix, the model was docked in the densities of the PhPINK1-AMP-PNP and pPhPINK1-ADP dimers, and nucleotides and Mg 2+ ions were fitted into the densities.Model building and refinement were then performed on all three models.Regions with disordered/ ambiguous densities were not modeled.Cryo-EM data collection and refinement statistics are provided in Table 1.

Generation of stable cell lines
HeLa PINK1 −/− cells stably expressing YFP-Parkin and HsPINK1 were generated by sequential introduction of YFP-Parkin followed by HsPINK1 into HeLa PINK1 −/− cells.YFP-Parkin was introduced using retroviral transduction with the pBMN-YFP-Parkin plasmid (gift from R. Youle; Addgene plasmid, 59416), followed by fluorescence sorting.HsPINK1 WT and the M318G mutant were introduced using lentiviral transduction with pFUP-HsPINK1 plasmids, followed by selection with puromycin.
Fractionation and ubiquitin phosphorylation assay Ubiquitin phosphorylation assays using heavy membrane-associated HsPINK1 from OA-treated HeLa PINK1 −/− cells transfected with HsPINK1 WT, M318A, and M318G were performed as described previously (12).HeLa PINK1 −/− cells (1 × 10 6 ) were seeded in 10-cm dishes.After 48 hours, cells were transfected with 5 μg of pcDNA5 d3 -HsPINK1 WT, M318A, or M318G using the Lipofectamine 3000 Transfection Reagent (Invitrogen).Twenty-four hours after transfection, HsPINK1 was stabilized by OA treatment for 2 hours.Cells were harvested by scraping in cold phosphate-buffered saline (PBS) and pelleted by centrifugation at 200g for 5 min at 4°C.Cells were permeabilized by incubating for 20 min at 4°C in 1 ml of fractionation buffer [20 mM Hepes (pH 7.4), 250 mM sucrose, 50 mM KCl, and 2.5 mM MgCl 2 ] supplemented with 0.025% (w/v) digitonin, 1× cOmplete Protease Inhibitor Cocktail (Roche), and 1× PhosSTOP (Roche).Heavy membrane fractions were pelleted by centrifugation at 14,000g for 5 min at 4°C, washed once with 1 ml of fractionation buffer and then resuspended in 100 μl of fractionation buffer.Ubiquitin (15 μM) was added, and membranes were divided into 50 μl of aliquots.The reaction was initiated with 1 mM ATP (Sigma-Aldrich), KTP (Biolog), or an equivalent volume of water and incubated at 30°C for 60 min or the indicated times with gentle agitation.Heavy membranes were pelleted by centrifugation at 14,000g for 5 min at 4°C, and samples for the ubiquitin containing supernatant and the HsPINK1 containing heavy membrane pellet were prepared in SDS sample buffer.Western blotting was performed as described above.

Immunofluorescence assay
HeLa PINK1 −/− cells stably expressing YFP-Parkin and HsPINK1 were seeded on HistoGrip-coated coverslips 48 hours before treatment with OA and/or 200 μM kinetin for the indicated times.Cells were then fixed with 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer on a rocker for 10 min, rinsed three times with PBS, and permeabilized with 0.1% (v/v) Triton X-100 in PBS for 10 min.After that, samples were blocked with 3% (v/v) goat serum in 0.1% (v/v) Triton X-100/PBS for 15 min and incubated with antigreen fluorescent protein (Thermo Fisher Scientific, A10262, RRID:AB_ 2534023) and anti-mitochondrial HSP60 (Abcam, ab128567, RRID:AB_ 11145464) antibodies for 90 min.Following three washes with PBS and subsequent 1 hour of incubation with Alexa Fluor 488 goat antichicken IgG (Thermo Fisher Scientific, A32931, RRID:AB_2762843) and Alexa Fluor 647 goat anti-mouse IgG (Thermo Fisher Scientific, A21235, RRID:AB_2535804), the coverslips were washed three times with PBS and mounted with a tris-buffered DABCO-glycerol mounting medium onto glass slides.Imaging of the coverslips was done with an inverted Leica SP8 confocal laser scanning microscope under 63×/1.40numerical aperture objective (Oil immersion, HC PLAPO, CS2; Leica microsystems).Details on the staining procedure are available at (49).

Supplementary Materials
This PDF file includes: Figs.S1 to S9 References

Fig. 1 .
Fig. 1.Determining nucleotide-bound PhPINK1 structures.(A) A 15-Å low-pass-filtered (lPF) cryo-eM density map of the published PhPinK1 d357A dodecamer [(12); eMd-25680].individual PhPinK1 monomers are shown in alternating colors.Accessible AtP binding sites are highlighted in the dotted outlines (two binding sites per enclosed outline).(B) Workflow to generate the wild-type (Wt) PhPinK1 dodecamer for cryo-eM analysis in complex with nucleotides.the illustrative micrograph image is reused from fig.S2c; see there for experimental detail.(C) Structures of the nucleotide-free, AMP-PnP-bound, and AdP-bound PhPinK1 dimer (only chain B is shown; see fig.S3 for whole dimers) at 2.75-, 2.84-, and 3.13-Å resolution, respectively.insertion-3 and the αc helix are colored in yellow and orange, respectively.density of AMP-PnP, AdP, and Mg 2+ in the PhPinK1 AtP binding site are shown as a mesh.aa, amino acids.dotted lines indicate regions lacking cryo-eM density.

Fig. 2 .
Fig. 2. PhPINK1-nucleotide interactions.(A) details of the interaction of AMP-PnP and AdP with the AtP binding site of PhPinK1 (chain B of each dimer).interacting residues are shown as sticks, and polar interactions are shown as dotted lines.the interaction with (phosphorylated) Ser 202 of chain A (gray) is also shown.(B) Superimpositions of the n-lobe of AMP-PnP-bound and AdP-bound PhPinK1 with nucleotide-free PhPinK1, revealing differences in conformation of the P-loop and insertion-3 (highlighted in the dotted outlines).lys 167 is highlighted in a lighter shade.(C) the n-lobe of the published phosphorylated and ubiquitin-bound PhPinK1 complex [Ub tvln, ubiquitin t66v l67n mutant; PdB: 6eQi; (20)], in the same orientation as in (B).lys 167 (highlighted in a darker shade) interacts with both the ordered insertion-3 and ubiquitin (gray).

Fig. 3 .
Fig. 3.The PINK1 gatekeeper residue prevents KTP binding.(A) chemical structures of AtP and KtP. the n 6 -furfuryl group of KtP is highlighted in red.(B) Melting temperatures of Wt PhPinK1 (residues 115 to 575) in the presence of AtP or KtP.experiment was performed three times in technical duplicates.(C) time course ubiquitin phosphorylation assay using Wt PhPinK1 in the presence of AtP or KtP and analyzed by Phos-tag SdS-polyacrylamide gel electrophoresis (PAGe).experiment was performed in triplicate.(D) time course ubiquitin phosphorylation assay using HsPinK1-containing heavy membranes from OA-treated hela cells in the presence of AtP or KtP (see Materials and Methods) and analyzed by Western blotting.experiment was performed in triplicate.(E) the AtP binding site of AMP-PnP-bound PhPinK1, revealing that the gatekeeper Met 290 would sterically block an n 6 -modified AtP analog from binding PhPinK1.(F) conservation of the PinK1 gatekeeper residue.Ph, Pediculus humanus corporis; Hs, Homo sapiens; Mm, Mus musculus; Rn, Rattus norvegicus, Dr, Danio rerio; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; Tc, Tribolium castaneum.(G) Melting temperatures of PhPinK1 Wt and gatekeeper mutants M290A and M290G in the presence of AtP or KtP.experiment was performed two times in technical duplicates, while M290A in the absence of nucleotide was performed three times in technical duplicates (see fig.S7).(H) Ubiquitin phosphorylation assay using PhPinK1 Wt and gatekeeper mutants M290A and M290G in the presence of AtP or KtP for 2 hours and analyzed by Phos-tag SdS-PAGe.experiment was performed in triplicate.(I) Ubiquitin phosphorylation assay using HsPinK1-containing heavy membranes from OA-treated hela cells in the presence of AtP or KtP for 2 hours (see Materials and Methods) and analyzed by Western blotting.experiment was performed in triplicate.

Fig. 5 .
Fig. 5. Kinetin-activated HsPINK1 M318G induces Parkin translocation.YFP-Parkin translocation in fixed hela PINK1 −/− cells stably expressing HsPinK1 Wt or M318G and immunostained for mthSP60 (mitochondrial marker).cells were treated with either OA alone or in combination of 200 μM kinetin for 1 hour before fixing and staining.While YFP-Parkin translocation was induced with OA alone in cells expressing HsPinK1 Wt, translocation in HsPinK1 M318G-expressing cells was only induced when cotreated with kinetin.however, the M318G mutant is not completely inactive; when cells were treated with OA alone for 2 hours, YFP-Parkin translocation was observable (see fig.S9).Scale bars, 20 μm (left three columns) and 10 μm (right three columns).White boxes (left) depict the zoomed-in area.experiment was performed in duplicate with fig.S9, and representative images are shown.YFP, yellow fluorescent protein.